1 School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, The University of Newcastle and Hunter Medical Research Institute, Room 411 Medical Sciences Building, University Drive, Newcastle, NSW 2308, Australia

2 Institute of Neuroscience & Psychology, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow, UK

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Background

Acute and chronic pain in axial structures, like the back and neck, are difficult
to treat, and have incidence as high as 15%. Surprisingly, most preclinical work on
pain mechanisms focuses on cutaneous structures in the limbs and animal models of
axial pain are not widely available. Accordingly, we developed a mouse model of acute
cervical muscle inflammation and assessed the functional properties of superficial
dorsal horn (SDH) neurons.

Results

Male C57/Bl6 mice (P24-P40) were deeply anaesthetised (urethane 2.2 g/kg i.p) and
the rectus capitis major muscle (RCM) injected with 40 μl of 2% carrageenan. Sham
animals received vehicle injection and controls remained anaesthetised for 2 hrs.
Mice in each group were sacrificed at 2 hrs for analysis. c-Fos staining was used
to determine the location of activated neurons. c-Fos labelling in carrageenan-injected
mice was concentrated within ipsilateral (87% and 63% of labelled neurons in C1 and
C2 segments, respectively) and contralateral laminae I - II with some expression in
lateral lamina V. c-Fos expression remained below detectable levels in control and
sham animals. In additional experiments, whole cell recordings were obtained from
visualised SDH neurons in transverse slices in the ipsilateral C1 and C2 spinal segments.
Resting membrane potential and input resistance were not altered. Mean spontaneous
EPSC amplitude was reduced by ~20% in neurons from carrageenan-injected mice versus
control and sham animals (20.63 ± 1.05 vs. 24.64 ± 0.91 and 25.87 ± 1.32 pA, respectively).
The amplitude (238 ± 33 vs. 494 ± 96 and 593 ± 167 pA) and inactivation time constant
(12.9 ± 1.5 vs. 22.1 ± 3.6 and 15.3 ± 1.4 ms) of the rapid A type potassium current
(IAr), the dominant subthreshold current in SDH neurons, were reduced in carrageenan-injected
mice.

Conclusions

Excitatory synaptic drive onto, and important intrinsic properties (i.e., IAr) within SDH neurons are reduced two hours after acute muscle inflammation. We propose
this time point represents an important transition period between peripheral and central
sensitisation with reduced excitatory drive providing an initial neuroprotective mechanism
during the early stages of the progression towards central sensitisation.

Keywords:

Mice; A-current; EPSC; Carrageenan; Action potential

Introduction

Acute inflammation in peripheral structures, such as muscle, results in nociceptor
activation and pain. Unfortunately, pain sometimes outlasts the initial inflammatory
insult and persists in a more chronic form. Long-term changes in components of the
pain neuroaxis such as altered synapses and neuronal properties are thought to underlie
this process [1]. Understanding the mechanisms involved in the progression from the acute to chronic
pain state, however, remains a major challenge in pain neurobiology.

Regardless of the site involved in chronic pain states, the central processing of
nociceptive signals begins in the superficial dorsal horn (SDH; laminae I-II) of the
spinal cord [2]. Here, the passage of information to be relayed from primary afferent fibres to higher
brain regions can be gated by synaptic inputs from local segmental interneurons and
descending brainstem centres. Such a process greatly influences whether nociceptor
activation can ultimately be perceived as pain [3]. Since the initial discovery that the electrophysiological properties of neurons
in the lumbar spinal cord could be altered after repeated noxious peripheral input
[4], plasticity in spinal cord pain circuits, termed central sensitisation, has been
considered a key element in the development of chronic pain [5,6].

Surprisingly, most of our understanding of the spinal mechanisms underlying plasticity
in pain circuits comes from studies on the rodent hindlimb following nerve injury
or inflammation, even though there is both clinical and pre-clinical evidence suggesting
that such mechanisms may differ for axial and limb pain [7]. Patients with neck pain often complain of a range of symptoms not obviously associated
with damage to neck structures. These include dizziness, visual disturbances, general
weakness, numbness or parathesis, cutaneous hyperalgesia, as well as psychological
symptoms such as disturbances in concentration and memory [8]. These signs and symptoms are not normally associated with limb pain where sensory
disturbances tend to be more localised [9]. At the cellular level we know that upper cervical SDH neurons receive input from
a unique combination of tissues including cutaneous and deep structures in the neck,
head and cranial vault [10,11]. This afferent convergence presumably plays an important role in the complex presentation
of pain originating in neck tissues [12]. Together these data suggest the mechanisms underlying processing of nociceptive
signals originating in neck structures differ from those in the limbs, and may be
important for the development of neck pain and its symptoms.

In order to begin to understand the spinal mechanisms that underlie the development
of chronic neck pain, we assessed the impact of acute muscle inflammation on the synaptic
and intrinsic properties of SDH neurons in the mouse upper cervical spinal cord. After
two hours of acute inflammation of the rectus capitus major (RCM) muscle we find excitatory
synaptic drive to SDH neurons and the rapid A-type potassium current are reduced in
SDH neurons. We hypothesise that two hours post inflammation is an important epoch
during the cascade of events where aberrant nociceptive signaling transitions from
a peripheral to central locus.

Methods

The University of Newcastle Animal Care & Ethics Committee approved all experimental
procedures. Male C57/Bl6 mice (P24 - P40) were deeply anaesthetised with urethane
(2.2 g/kg i.p) and underwent the following treatments: control animals remained anaesthetised
for 2 hrs; sham animals received a unilateral injection of 40 μl phosphate-buffered
saline (PBS, pH 7.4) via a 26 gauge needle into the RCM muscle; experimental animals
received an injection of 2% carrageenan (in phosphate buffered saline - PBS) into
the RCM. This muscle is particularly large in rodents and runs from the spinous process
of C2 to the skull. It is easily located by palpating the external occipital protuberance
(inion) and the spinous process of the C7 vertebrae. The location of the C1 spinous
process in the mouse is one-fifth the distance between the inion and the C7 spinous
process. The needle was inserted just lateral to the midline at the level of C1 (angled
~45° to the horizontal). The needle was inserted until it reached the occiput and
withdrawn so the tip remained in the RCM muscle belly. Mice were maintained under
anaesthetic for 2 hours prior to preparation for immunohistochemistry or electrophysiology.

Immunohistochemistry

After 2 hours of urethane anaesthesia animals were further injected with Ketamine
(100 mg/kg), and perfused through the ascending aorta with PBS (20 ml), followed by
20 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The cervical
spinal cord (obex - C5) was removed and post-fixed in 4% paraformaldehyde (2 hours)
prior to washing with PB and cryoprotection in PB containing 30% sucrose at 4°C for
48 hrs. Serial transverse sections (60 μm) were cut and every 5th section was processed
for Fos-like immunoreactivity.

Sections from carrageenan-injected (n = 3), sham 128 (PBS; n = 3), and control (uninjected;
n = 4) animals were incubated in 50% ethanol for 30 minutes to enhance antibody penetration
and then incubated in 0.03% hydrogen peroxide in PB for 20 minutes to block endogenous
peroxide activity. Sections were then incubated for 72 hours with a polyclonal goat
anti-fos antibody (diluted 1:1000 in PBS; Santa Cruz Biotechnology, Santa Cruz, CA,
USA), followed by a biotinylated donkey anti-goat IgG secondary antibody (1:500; Jackson
Immunoresearch, West Grove, PA, USA) for an additional 24 hours. Sections were then
incubated in ExtrAvidin-peroxidase (diluted 1:1000. Sigma-Aldrich; Cat no. E2886)
and peroxidase labeling visualised using 3′3′-diamino-benzadine (DAB) as a chromagen.
All antibodies and the peroxidase-labelled ExtrAvidin were diluted in PBS containing
0.3% Triton X-100. Sections were then dehydrated, cleared and mounted in serial order
on slides. The relative position of all c-Fos positive cells within the C1 and C2
segments were plotted onto templates of the appropriate spinal segment (adapted from
[13]) using Adobe Illustrator CS4 (Adobe Systems Inc., San Jose, CA, USA). Mean number
of c-Fos positive cells per spinal segment was calculated from these data.

Electrophysiology

Sham (n = 15 mice) and carrageenan injected mice (n = 10) were decapitated as close
as possible to the thorax while still under urethane anaesthesia. Control animals
(uninjected; n = 16 mice) were anaesthetised with ketamine (100 mg/kg i.p) before
decapitation. The cervical vertebral column was isolated and immersed in ice-cold
oxygenated sucrose substituted artificial cerebrospinal fluid (s-ACSF) containing
(in mM): 250 sucrose, 25 NaHCO3, 10 glucose, 2.5 KCl, 1 NaH2PO4, 1 MgCl2, 2.5 CaCl2. The spinal cord was removed and a shallow cut made down the contralateral side of
the spinal cord (ventral surface) to identify the ipsilateral side for recording.
Transverse slices (300 μm thick) were obtained from the C1-C2 region using a vibratome
(Leica VT-1000S; Leica Microsystems, Wetzlar, Germany). All slices were transferred
to an interface storage chamber containing oxygenated ACSF (118 mM NaCl substituted
for sucrose in s-ACSF), and allowed to equilibrate for 1 hr at room temperature (approximately
22-24°C), prior to recording.

The whole cell recording configuration was first established in voltage clamp (holding
potential −60 mV). Series resistance (Rs), input resistance (RIN), and capacitance were measured from the averaged response (5 repetitions) to a 5 mV
hyperpolarising voltage step. These parameters were observed following each protocol,
and data were rejected if values changed by >20%. Initially, spontaneous excitatory
post-synaptic currents (sEPSCs) were recorded for each neuron in voltage-clamp (holding
potential −60 mV; 60 s duration). The recording mode was then switched to current
clamp. The membrane potential recorded following this switch (~15 s later) was designated
resting membrane potential (RMP). A series of depolarising ‘step-current’ injections
(20 pA increments; 800 ms, delivered every 8 s) were then injected in order to study
action potential (AP) discharge in each recorded neuron. Finally, the recording configuration
was returned to voltage clamp and a protocol was run to study voltage-gated subthreshold
ionic currents. This protocol first hyperpolarised the membrane potential to −90 mV
(for 1 s) before delivering a depolarising step to −40 mV (for 200 ms) before returning
to −60 mV. All membrane currents were filtered at 2 kHz and stored on a Macintosh
G4 computer using Axograph v.10 (Axon Instruments, Foster City, CA, USA) for future
analysis.

Data analysis

All data were analysed off-line using semi-automated procedures within Axograph software.
sEPSCs were detected and captured using a scaled template method and subsequently
inspected [14]. Some events were rejected if they overlapped or did not include a stable baseline
before the rising phase of the captured event. For each recording, stationary plots
of sEPSC interevent interval were constructed for the duration of the analysis period.
These plots were inspected to ensure no time dependent change or heavy clustering
of data points, which would both be indicative of burst activity, were included in
the analysis. Several sEPSC parameters were measured including mean amplitude, rise
time, decay time and instantaneous frequency. Individual APs evoked during step current
injection were detected and captured using the derivative threshold method for spike
initiation (set at dV/dt ≥ 20 Vs−1). The pattern of AP discharge was classified into categories using previously described
criteria [15,16] as; single spiking, initial bursting, tonic firing, delayed firing, or reluctant
firing. Likewise, subthreshold currents (IAr, IAs, T-type calcium, Ih, & mixed) were classified and compared between control, sham and experimental (carrageenan-injected)
animals.

Statistical analysis

All statistical analyses were carried out using SPSS statistics software v.18.0 (SPSS
Inc., Chicago, IL, USA). One-way ANOVA coupled with Student-Newman-Keuls and Scheffe
post-hoc tests were used to compare variables from control, sham and experimental
animals. Data with unequal variances were compared using the non-parametric Kruskal-Wallis
test. Chi-square tests were utilised to determine whether the prevalence of discharge
categories and subthreshold currents differed between the groups. Statistical significance
was set at p < 0.05. All values are presented as the mean ± S.E.M.

Results

Experiments were designed to characterise the early events that occur in SDH neurons
following acute neck muscle inflammation. To establish the segmental level and distribution
of activated neurons, spinal cords were processed to visualise the immediate early
gene product c-Fos (Figure 1). The level of neuronal activation (determined by number of c-Fos positive profiles)
was assessed in the C1-C2 spinal segments of control, sham, and carrageenan-injected
animals.

Whole cell patch-clamp recordings were obtained from 205 SDH neurons in 41 animals
from carrageenan-injected (n = 58 cells from 10 mice), sham (n = 68 cells from 15
mice), and control (n = 79 cells from 16 mice) animals. These experiments examined
a range of electrophysiological properties known to contribute to neuronal excitability
and plasticity. The location of recorded neurons was documented in latter experiments
(after image capture software) to ensure similar sampling occurred throughout the
SDH under all experimental conditions. Figure 2 summarises the results of this analysis and confirms that similar regions of the
upper cervical SDH were sampled in carrageenan-injected, sham and control mice (n = 33,
39, and 36 neurons, respectively).

Figure 2.Location of recorded neurons in spinal cord slices. The location of recorded neurons was documented to check and account for bias. Low
magnification images (5×) were captured at the conclusion of recording sessions with
the recording pipette still in place. Recording locations were then plotted on a standardised
transverse template of the upper cervical spinal cord. Recording locations were then
consolidated onto a single template for control, sham, and carrageenan-injected experiments.
Comparison of these maps indicates similar sampling in both mediolateral and dorsoventral
planes for each experimental condition.

In a subset of recordings (n = 48, 44, 58; control, sham and carrageenan-injected,
respectively) we examined AP discharge, evoked by step current injection, to assess
whether acute neck muscle inflammation altered the intrinsic properties (and excitability)
of upper cervical SDH neurons. The features of voltage responses exhibited at multiple
current step injections were used to classify AP discharge patterns into five categories,
as previously described [15,16]. Single spiking neurons, discharged one AP at the beginning of step current injection
regardless of current intensity; initial bursting neurons discharged a brief, adapting
train of APs limited to the beginning of the current step; tonic firing neurons exhibited
sustained discharge for the duration of the current step; delayed firing neurons discharged
APs after a delay from step current onset; and reluctant firing neurons failed to
fire APs in response to any current step, independent of intensity. All discharge
patterns were observed under the three experimental conditions (Figure 4A), and there was no significant difference in the proportion of these discharge categories
in control, sham and carrageenan-injected animals (Figure 4B, χ2; p = 0.27).

Figure 4.Acute neck muscle inflammation does not alter action potential discharge properties. A. Dorsal horn neurons can be classified by the temporal features of their action potential
discharge. We have previously described five AP discharge patterns under in vitro
[16] and in vivo conditions [15], and also across the lumbar, thoracic and cervical spinal cord in adult mice [17]. Traces show representative examples of each AP discharge pattern (Tonic Firing -
TF, Initial Bursting - IB, Delayed Firing - DF, Single Spiking - SS, and Reluctant
Firing RF) recorded from control (left), sham (middle), and carrageenan-injected animals
(right). Horizontal bars denote −50 mV membrane potential. B. The incidence of the five AP discharge patterns was similar in control, sham, and
carrageenan-injected animals.

In some neurons we also tested for the presence of the four main subthreshold currents
that shape AP discharge in SDH neurons (n = 38, 39, 41; carrageenan-injected, sham,
and control, respectively, Figure 5). This analysis employed a voltage-step protocol to identify different subthreshold
currents in adult mouse SDH neurons [18]. These include two types of outward potassium current that are activated by a depolarising
step (−90 to −40 mV). The first displays fast activation and inactivation kinetics
and is termed a rapid A-type current (IAr), while a second exhibits slower activation/inactivation kinetics and is termed a
slow A-type current (IAs). These two currents are easily differentiated as the rapid form (IAr) was fully inactivated during a 200 ms activation step (to −90 to −40 mV), whereas
the slower form did not fully inactivate over the same period. A criterion threshold
of 50 ms for the outward current inactivation time constant was used to classify currents
as either IAr (<50 ms) or IAs (>50 ms). This approach resulted in two non-overlapping distributions in recordings
from control, sham and carrageenan-injected mice. The other major subthreshold current
types are inward currents. The first is an inward current with rapid activation/inactivation
kinetics (in response to a −90 mV to −40 mV step), consistent with the low threshold
‘T-type calcium current’ (ICa; Figure 5A). The second, inward current is activated by hyperpolarisation (−60 mV to −90 mV
step) and exhibits slow activation kinetics consistent with the well-described current
termed Ih (Figure 5A). Importantly, coexpression of the currents observed using our protocol was not
frequently observed. For example, a combination of ICa and Ih occurred in <10% of the neurons sampled (n = 4/41, 2/39, and 2/38, control, sham
and carrageenan-injected respectively). Co-expression of other currents was not directly
assessed in these experiments, as this would have required pharmacological analysis,
and limited the collection of data that was the principal focus of our experiments.
Previous work using pharmacological analysis, albeit in lumbar SDH neurons, suggests
a single subthreshold current typically dominates in response to our protocol [18]. We do not discount the possibility, however, that combinations of IAr, IAs, and ICa may exist in some of these neurons or that the incidence of mixed currents is higher
in upper cervical SDH neurons.

Figure 5.Acute neck muscle inflammation alters subthreshold voltage-activated currents. A. Traces show representative voltage-activated current recordings (from control, sham,
and carrageenan-injected animals) in response to voltage step protocol (bottom black
traces, note different scale for lower right Ih currents). We have previously shown this protocol identifies one of four dominant
currents in dorsal horn neurons: rapid A-type potassium currents (IA rapid); slow A-type potassium currents (IA slow); T-type calcium currents (ICa); and a hyperpolarisation-activated non-selective cation current (Ih). Analysis of these currents across the three experimental conditions showed the
characteristics of IA slow, ICa, or Ih did not differ under the three conditions. In contrast, the amplitude of the IA rapid current was reduced in carrageenan-injected animals (see text). B. Plots show the incidence of sub-threshold voltage activated current types was similar
in control, sham, and carrageenan-injected animals.

Discussion

We assessed the impact of two hours of acute neck muscle inflammation on the properties
of SDH neurons in the upper cervical spinal cord. Our c-Fos experiments showed carrageenan-induced
muscle inflammation produced neuronal activation in both ipsilateral and contralateral
laminae I – II, and to a lesser extent in lamina V of the C1 and C2 spinal segments.
These findings confirmed that neurons in laminae I - II should be the principal target
in subsequent electrophysiological recordings from acutely prepared spinal cord slices.

Assessment of c-Fos expression, the protein product of the proto-oncogene c-fos, is a well-established method for determining neuronal activation after noxious peripheral
stimulation [19,20]. In our study, c-Fos expression occurred in neurons located predominantly in laminae
I - II. This matches the terminal field distribution of nociceptive afferent fibres
[2], and the location of c-Fos expressing neurons after application of noxious peripheral
stimuli [20]. Some c-Fos labelling was also observed in deeper layers (lateral lamina V) of the
dorsal horn. These superficial and deep locations closely match those noted in other
rodent injury models involving deep tissues, such as muscle. For example in rat, chronic
constriction injury of the sciatic nerve [21], adjuvant-induced arthritis [22], and nerve growth factor injection into semispinal neck muscles [23] resulted in c-Fos labelling in laminae I-II and V-VII. In contrast, capsaicin injection
into the trapezius and splenius muscles of cats does not label many neurons in lamina
II [24]. Thus, some differences exist in the extent to which neurons in laminae II are involved
in processing noxious input from muscle across species.

No labelling was observed in control or sham (saline injection) animals in our c-Fos
experiments. These data suggest neither handling nor the manipulation of muscle tissue
associated with the injection protocol resulted in significant activation of dorsal
horn interneurons. Similar results have been observed for limb muscles when c-Fos
expression has been compared with that in vehicle-injected animals [25]. Our unilateral carrageenan injections resulted in c-Fos labelling in both ipsilateral
and contralateral superficial laminae (I-II) with some labelling in deeper laminae.
Bilateral c-Fos expression has been observed after capsaicin injection into other
neck muscles (trapezius and splenius capitis) in cats [24]. This contrasts with the data for limb muscles where inflammation produces neuronal
activation that is confined to the ipsilateral side of the spinal cord. Thus, the
major pattern of c-Fos activation we described following carrageenan injection into
RCM, fits with activation of muscle nociceptive pathways in axial musculature. Thus,
recording from neurons in ipsilateral laminae I-II should detect inflammation-related
changes in the spinal dorsal horn.

In our electrophysiological analysis of SDH neurons in spinal cord slices prepared
two hours after RCM inflammation we observed two major changes: i) AMPA-mediated excitatory
drive decreased and ii) the amplitude and kinetics of the rapid, but not slow IA type potassium current were altered. Assuming acute neck muscle inflammation enhanced
nociceptive signaling (cf. our c-Fos results above), it is surprising that we found excitatory drive to SDH
neurons was reduced (as assessed by sEPSC amplitude and charge). It is noteworthy,
however, that other studies have also described reduced excitatory drive to SDH neurons
in pain models, specifically in GAD67 positive inhibitory interneurons [26]. In these experiments the frequency of spontaneous excitatory input, rather than
amplitude, was reduced in neuropathic animals. The authors proposed that reduced excitatory
drive to inhibitory interneurons, when placed in the context of nociceptive signaling,
would reduce inhibitory drive in the SDH and contribute to hyperalgesia. While our
data does not allow us to identify recorded neurons as excitatory or inhibitory, similar
plasticity in the form of reduced sEPSCs onto inhibitory interneurons would contribute
to enhanced nociceptive signaling in acute neck muscle inflammation.

Regardless of the identity of neurons that undergo reduced excitatory drive in our
model, numerous studies have confirmed that inflammation alters the expression of
AMPA type glutamate receptors. For example, expression of calcium permeable AMPA receptors
containing GluR1 subunits has been shown to increase, whereas expression of calcium
impermeable GluR2 containing subunits was reduced [27-31]. Importantly, all these studies assessed receptor expression 24 hours after peripheral
inflammation. In contrast, our data showing sEPSC kinetics were unaltered in carrageenan-injected
recordings imply that AMPA receptor expression is unchanged at the two-hour time point
used following axial muscle inflammation. Phosphorylation of GluR1 and GluR2 has,
however, been demonstrated at time points commensurate with those in our study [32]. This would enhance excitatory drive in SDH neurons. Importantly, most GluR1/2 subunit
expression and phosphorylation studies have used biomolecular techniques in spinal
cord homogenates where synaptic and extrasynaptic receptors and the precise laminae
location of neurons are not known. Thus our experiments, which assessed synaptic receptor
function in specific laminae, suggest levels of GluR plasticity vary according to
time after inflammatory insult.

Several electrophysiological studies have examined the properties of primary afferent
synapses in the SDH after peripheral inflammation, and report that primary afferent
synaptic function was generally enhanced [33,34]. Again this contrasts with the reduced excitatory drive that we observed. However,
our sEPSCs recordings will have included excitatory currents arising from local interneurons,
descending systems, as well as primary afferents. Relevant to this point, previous
work has shown that ablation of afferent input by dorsal rhizotomy, or selective removal
of peptidergic afferents with neonatal capsacin treatment, does not reduce mEPSC frequency
recorded from SDH neurons in spinal slices [35,36]. This suggests that the majority of sEPSCs recorded in an isolated spinal slice such
as that in our experiments, come from local interneurons and not primary afferent
terminals. Thus, any change to primary afferent synapses may have gone undetected
in our experiments.

When sEPSCs have been recorded in the SDH in peripheral inflammation models it appears
their properties are not altered within the first 24 hours after inflammation [37,38]. Interestingly, the Li et al. study showed that sEPSC frequency was enhanced when
inflammatory insults were delivered to neonates but not in animals older than P14.
The most likely explanation for the reduced sEPSC amplitudes we observed comes from
work that has assessed the contribution of the initial primary afferent barrage and
its time course during an inflammatory insult. For example, peripheral nerve block
with lignocaine during muscle inflammation prevents the development of plasticity
(or central sensitisation) in spinal neurons, whereas nerve block outside the first
two hours of inflammation does not protect against central sensitisation [39]. This finding suggests primary afferent input within the first two hours of inflammation
plays a crucial role in establishing central sensitisation. In our experiments, the
barrage of primary afferent input would be expected to be the principle driver of
central sensitisation, which is reflected as the expression of c-Fos in certain neuronal
populations. The fact, however, that we did not observe an enhancement of sEPSC frequency
or amplitude may partly be explained by the in vitro slice preparation we used. Specifically,
previous work has shown that the removal of populations of primary afferent inputs
either by dorsal rhizotomy or neonatal capsacin does not affect the properties of
sEPSCs recorded in slices [35,38]. This implies that most sEPSCs recorded in slices originate from local interneurons
rather than primary afferents. Further support of our interpretation comes from work
in an in vitro hemisected spinal cord preparation where inflammation-induced central
sensitisation was only detected six hours after inflammation in the absence of peripheral
input [34]. These data, when combined with our finding that excitatory drive is decreased two
hours after acute muscle inflammation, suggests a complex sequence of events occurs
over the onset, establishment and maintenance of inflammatory pain and associated
central sensitisation.

The reduced excitatory drive we observed two hours after acute neck muscle activation
was accompanied by a reduction in the amplitude and time course of the IAr - type potassium current. IAr currents are expressed widely in the CNS and regulate AP discharge and neuronal output.
In the dorsal horn, IAr currents are thought to play an important role in reducing neuronal excitability.
For example, IAr currents are preferentially associated with the delayed firing pattern of AP discharge
(Figure 4A). Likewise, SDH neurons expressing delayed firing and IAr currents exhibit reduced AP discharge compared to tonic firing and initial bursting
neurons when activated by current protocols they are likely to receive in vivo (synaptic
vs. square step current injection) [40]. Furthermore, a number of studies have suggested IAr expression is a feature of excitatory interneurons [41,42]. This suggests that diminished IAr would substantially increase excitability and nociceptive signaling in the dorsal
horn. Thus, any intervention that reduces IAr current in SDH neurons leads to enhanced excitability.

Comparable modulation of IAr in the dorsal horn has been reported after peripheral inflammation. For example,
enhanced excitability in dorsal horn neurons is observed as early as one hour after
carrageenan injection into the hindpaw of young (P7-12) mice [43]. A detailed analysis of IAr potassium currents in these animals identified a shift in the current’s steady state
inactivation. The authors suggested this would act like “a relaxation of the brake
on excitability at physiologically meaningful potentials”. Interestingly, we have
previously demonstrated the converse (i.e., enhanced braking) in spastic mice with
glycine receptor mutations [18]. In this case, modulation of IAr was seen as a compensatory mechanism to enhance neuronal inhibition and maintain
stable sensory processing. Together, this work suggests IAr can be strongly modulated to maintain an excitability set-point in the SDH. Importantly,
a signalling pathway involving metabotropic glutamate receptor activation and extracellular
signal-regulated kinase dependent phosphorylation of Kv4.2 channels has been shown
to regulate IAr currents in SDH neurons [44,45]. Furthermore, this work demonstrated that inflammation of peripheral tissues can
activate this pathway and reduce IAr. This provides a potential mechanism for our observations during acute neck muscle
inflammation.

Given IAr currents are diminished in SDH neurons from carrageenan-injected animals one might
expect this to impact on neurons with the delayed- and reluctant firing AP discharge
pattern as the two have been associated in a number of previous studies [18,46,47]. For example, diminished IAr would either reduce the delay before AP discharge or reduce the proportion of neurons
exhibiting delayed and reluctant firing and convert them to more excitable forms of
discharge such as tonic firing or initial bursting. There was, however, no difference
in the incidence of AP discharge patterns across the three conditions: control, sham
and carrageenan-injected. This finding may be explained, at least partly, by the biophysical
properties of IAr currents, which are inactivated at depolarised membrane potentials [16,47]. The membrane potential of neurons in our recordings was approximately −60 mV across
all three conditions. At this potential much of IAr (~80 - 90%) is inactivated and therefore not able to reduce or delay AP discharge.
Under in vivo conditions, however, when membrane potential is fluctuating due to the
combination of inhibitory and excitatory synaptic input, IAr currents could have a greater impact on neuronal output. Thus, under in vivo conditions
the reduction in IAr we observed could enhance excitation and disrupt normal sensory processing.

Ultimately, our findings must be placed in the context of the whole animal. Fortunately,
relevant data are available on the time course of pain related behaviours following
carrageenan injections. Most of this work has involved carrageenan injection into
the plantar surface of the hindpaw [48-52] (rather than muscle). Carrageenan causes rapid edema, which peaks 3–4 hours post
injection and largely resolves by 24–72 hours [53]. Behaviourally, carrageenan’s capacity to induce thermal and mechanical hyperalgesia
is highly reproducible. These indices of altered pain sensation have been detected
as early as 15 minutes after carrageenan injection [48], though typically studies report effects to be fully developed at 1–4 hours [52]. Following onset, some variability exists in duration of the carrageenan-induced
mechanical and thermal hyperalgeisa. This variability might reflect the carrageenan
concentration (typically 1-3% is injected) as well as the species studied. However
all work suggests mechanical and thermal hyperalgeisa persists for at least 24 hours
[49]. The few studies that have injected carrageenan into muscle also support a rapid
onset for mechanical and thermal hyperalgesia, which is fully developed by two hours
and can last as long as 4–8 weeks [54,55]. Thus our study has described a number of central changes to both the synaptic and
intrinsic membrane properties of SDH neurons that equate to a relatively early time
point in the carrageenan-induced inflammatory pain model.

In summary, the present study suggests two hours of acute neck muscle inflammation
represents a transition point between the involvements of peripheral and/or central
sensitisation. As our study was undertaken at one time point post inflammation it
is difficult to predict where each of the alterations we observed lies in terms of
the train of events that leads to central sensitisation. The surprising result that
sEPSC amplitude and charge are reduced, suggest some excitatory inputs are depressed
in the hours following inflammation. This may reduce local excitatory drive in the
face of the initial excitatory barrage arising from the periphery. Alternatively,
if the neurons that experience a reduction in excitatory drive were inhibitory, an
associated reduction in the activity of this population would reduce inhibition in
the SDH and contribute to enhanced nociceptive signaling. It will be important in
future studies to assess whether our observation persists after peripheral drive abates
following the resolution of inflammation in the periphery. The diminished IAr-current after two hours of acute inflammation is, however, consistent with inflammation-induced
hyperexcitability in the spinal cord dorsal horn. Furthermore, there is a clear signalling
pathway that links hyperexcitation (and neuronal depolarisation), with ERK kinase-dependent
down regulation of A-currents [56]. Thus, the early barrage of afferent input from inflamed muscle could underpin this
observation. Our findings when combined with the existing literature on altered spinal
cord processing, albeit at longer times after acute insult, suggest a complex sequence
of events occurs in the dorsal horn following neck muscle inflammation. A greater
understanding of the order and duration of these changes may help to uncover new strategies
for blocking the transition from peripheral to central sensitisation.

Abbreviations

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

BMH carried out all surgery and electrophysiological recording, and helped draft the
manuscript. DIH carried out c-Fos staining. BMH and BAG analysed the electrophysiological
and anatomical datasets. BAG, PSB and RJC conceived the study, participated in its
coordination and drafted the final version of the manuscript. All authors have read
and approved the final version of the manuscript.

Acknowledgements

This work was supported by the National Health and Medical Research Council of Australia
(project grant 569206 and 631000), The Biotechnology and Biological Science Research
Council (Grant BB/J000620/1), The Hunter Medical Research Institute, and The University
of Newcastle.

Hedo G, Laird JMA, Lopez-Garcia JA: Time-course of spinal sensitization following carrageenan-induced inflammation in
the young rat: a comparative electrophysiological and behavioural study in vitro and
in vivo.